High Gravity and High Cell Density Enhance the Fermentation of Hexose Sugars
Present in Softwood Hydrolysates
Nuwan S. Kapu1, Maya Piddocke2, and Jack Saddler3
Forest Products Biotechnology and Bioenergy Research Group
Department of Wood Science, Faculty of Forestry, University of British Columbia
[email protected]; [email protected]; [email protected]. Website: www.bioenergy.ubc.ca
FOREST PRODUCTS
BIOTECHNOLOGY/BIOENERGY GROUP
Abstract
Biomass-based ethanol has the potential to provide substantial environmental, energy security and geopolitical benefits in comparison to fossil based
transportation fuels. However, fermentation of softwood derived sugars to ethanol is problematic due to the generally low sugar concentrations that can
be supplied and due to the naturally occurring and process derived inhibitors that are typically present. In the present study, the hexose rich, water soluble
fraction obtained after steam treatment of Douglas fir chips was supplemented with glucose up to 22% (v/v) to simulate high solids, un-detoxified
substrate, to see if a high sugar concentration/high cell density approach could better cope with known inhibitory materials. The addition of supplemental
glucose enabled the faster and quantitatively higher removal of hydroxymethylfurfural (HMF) and this observed boosting effect was more pronounced with
three superior strains. It appears that high cell density can provide effective fermentation at high sugar concentrations while enhancing inhibitor reduction.
A 77% ethanol yield could be achieved with strain Lallemand 4 after 48 hours at high cell density with some nutritional supplementation.
1. Introduction
5. Key Results
In contrast to sugar and starch
based ethanol, the sugars in
lignocellulosic
feedstocks
are
present as complex polymers (1,2).
Thus, lignocellulosic ethanol faces
four primary challenges:
• Without glucose supplementation
(ncbi.nlm.nih.gov)
 While low cells density resulted in
poor fermentation performance, for
most strains, high cell density
resulted in > 70% ethanol yield in 48
hours (Fig. 2).
Figure 2. Ethanol production by the studied strains in
glucose supplemented WSF (G-WSF) at low and high
cell density.
 prevalence of both pentose and
hexose sugars
 hydrolysates with low sugar
concentrations
 high levels of fermentation
inhibitors.
Figure 1. Multi-stress environment encountered by the yeast
in lignocellulosic ethanol production.
2. Fermentation of lignocellulosic feedstock: challenges
 Low sugar concentration in the liquor after pretreatment and hydrolysis steps
leading to low ethanol titer.
 Presence of naturally occurring and process derived (pretreatment through
fermentation) inhibitors.
3. Objectives
To determine whether a combined high cell density-high sugar concentration
approach can be used successfully to overcome the effect of fermentation
inhibitors on yeast metabolism to improve ethanol yield and productivity from
softwood hydrolysates.
4. Fermentation strategy
Substrate
 Low sugar concentration:
water soluble fraction (WSF) generated
by steam pretreatment of Douglas-fir with
3.17% total monomeric sugars content
 High sugar concentration:
simulated with glucose supplementation
of the original WSF to reach 22% total
monomeric sugars
Cell density
 Low cell density: 6x106 cells/ml
 High cell density:150x106 cells/ml
 With high cell density, the best
performing strains L4 and T1
resulted in ~98% yield with
complete glucose and mannose
consumption after 8 h and 4 h,
respectively (Fig. 3).
• With glucose supplementation
 With added glucose (G-WSF), strain
L4 at high cell density performed the
best, resulting in 65% ethanol yield
in 48 hours.
 Further nutrient supplementation for
strain L4 increased the yeast growth
rate by 25% and achieved an
ethanol yield of 77% (% of
theoretical maximum).
Glucose supplementation improved
inhibitor removal
 At high cell density, more then 98% of
HMF in the glucose supplemented
fermentations were metabolized within
24 hours by strain L4 (Fig. 4).
 At high cell density, furfural was nearly
completely converted for both the
glucose supplemented and the original
WSFs.
Yeast strains
 Lallemand: L1, L2, L3, L4
 Tembec: T1, T2
 Inhibitors concentration:
hydroxymethylfurfural (HMF) 2.4 g/L
furfural 0.5 g/L
acetic acid 3.0 g/L
total phenolics 1.7 g/L
References:
[1] Almeida et al.2007. Increased tolerance and conversion of inhibitors in lignocellulosic hydrolysates by Saccharomyces cerevisiae. J Chem Technol Biotechnol 82:340–349
[2] Humbird et al., 2011. Process design and economics for biochemical conversion of lignocellulosic biomass to ethanol. Technical report NREL/TP-5100-47764
Figure 3. Fermentation profile of strain L4 at high cell
density.
2.5
Concentration of HMF
(g/L)
 need for complex pretreatment
and enzymatic hydrolysis steps
to obtain fermentable sugars
High cell density improved the
fermentation performance
O-WSF (Original WSF)
G-WSF (Glucose supplemented WSF)
2
1.5
1
0.5
0
0
12
24
36
48
60
Fermentation time (h)
Figure 4. HMF removal by strain L4 at high cell density
from the original and glucose supplemented WSF.
0 hours
48 hours
Figure 5. Colour change potentially due to inhibitor
uptake by strain L4 after 48 fermentation hours at high
cell density and high sugar concentration.
6. Conclusions
High cell density is an effective strategy to ferment steam-pretreated Douglas-fir
hydrolysates to improve sugar consumption and obtain high ethanol yields.
Nutrient supplementation in addition to high sugar concentration and high cell density
further improved the ethanol yield.
Higher initial glucose concentration together with high cell density enables faster and
quantitatively higher removal of hydroxymethylfurfural (HMF) and furfural.